Persubstituted Cyclodextrin-Based Glycoclusters as Inhibitors ofProtein-Carbohydrate Recognition Using Purified Plant andMammalian Lectins and Wild-Type and Lectin-Gene-TransfectedTumor Cells as Targets†

Sabine Andre,*,‡ Herbert Kaltner,‡ Tetsuya Furuike,§ Shin-Ichiro Nishimura,*,| andHans-Joachim Gabius‡

Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich,Veterinarstr. 13, D-80539 Munich, Germany, and Laboratory for Glycocluster Project, Japan BioindustryAssociation, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, and Division ofBiological Sciences, Graduate School of Science, Hokkaido University, Kita-10 Nishi-8, Kitaku,Sapporo 060-0810, Japan. Received May 1, 2003; Revised Manuscript Received October 15, 2003

Multivalent glycoclusters have the potential to become pharmaceuticals by virtue of their targetspecificity toward clinically relevant sugar receptors. Their application can also provide fundamentalinsights into the impact of two spatial factors on binding, i.e., topologies of ligand (branching mode,cluster presentation) and carbohydrate recognition domains in lectins. Persubstituted macrocyclesderived from nucleophilic substitution of iodide from heptakis 6-deoxy-6-iodo-â-cyclodextrin by theunprotected sodium thiolate of 3-(3-thioacetyl propionamido)propyl glycosides (galactose, lactose andN-acetyllactosamine) were prepared. The produced glycoclusters were first tested as competitiveinhibitors in solid-phase assays. A plant toxin from mistletoe and an immunoglobulin G fraction fromhuman serum were markedly susceptible. A nearly 400-fold increase in inhibitory potency of eachgalactose moiety in the heptavalent form relative to free lactose (217-fold relative to free galactose)was detected. Thus, these glycoclusters can efficiently interfere, for example, with xenoantigen-dependent hyperacute rejection. Among the tested galectins selected from this family of adhesion-and growth-regulatory endogenous lectins, the substituted â-cyclodextrins acted as sensors to delineatetopological differences between the two dimeric prototype proteins. The relatively strong reactivitywith chimera-type galectin-3, a mediator of tumor metastasis, disclosed selectivity for glycoclusterbinding among galectins. Equally important, the geometry of ligand display (maxiclusters, bi- ortriantennary N-glycans) made its mark on the inhibitory potency. To further determine the sensitivityof a distinct galectin presented on the cell surface and not in solution, we established a stablytransfected tumor cell clone. We detected a significant response to presence of the multivalent inhibitor.This type of chemical scaffold with favorable pharmacologic properties might thus be exploited forthe design of galectin- and ligand-type-selective glycoclusters.

INTRODUCTION

The emergence of the concept of the sugar code entailsan increasing interest in the activities of endogenouslectins (Winterburn and Phelps, 1972; Laine, 1997;Reuter and Gabius, 1999; Rudiger et al., 2000; Gabiuset al., 2002; Kilpatrick, 2002). On account of cell surfacepresentation and capacity for endocytosis, medical ap-plications are being examined for several C-type lectins(Gabius, 1991, 1997; Rice, 1997; Yamazaki et al., 2000,2001; Davis and Robinson, 2002), by exploiting glycoli-gands as a postal code in vectorized drug delivery. Actingas role models for this purpose, the hepatic and mac-rophage asialoglycoprotein receptors and the tandem-repeat mannose macrophage receptor have already served

as proof-of-principle. In fact, the design of multivalentligands matching the rather rigid topological display ofthese lectins’ carbohydrate recognition domains was thebasis for achieving astounding affinity enhancements ofcustom-made glycoclusters relative to their monovalentform, termed the glycoside cluster effect (Lee and Lee,1994; Biessen et al., 1996; Grandjean et al., 2001; Eastand Isacke, 2002; Weigel and Yik, 2002). A similarlyimpressive example for how rational design of the liganddisplay accomplishes complementarity to fixed geometryof receptor sites is afforded by starfish and modularlyprepared pentavalent inhibitors for bacterial AB5 toxins(Schengrund, 2003). Thus, there is already ample reasonto conclude that the combination of synthetic chemistrywith lectin research promises insights into receptorfunctionality and a clinical perspective (Lee and Lee,1994; Bovin and Gabius, 1995; Roy, 1996, 2002; Kiessling

* To whom correspondence should be addressed. S.A.: Fax:+49-89-2180-2508; e-mail: Sabine.Andre@lmu.de. S.N.: Fax:+81-11-706-3435; e-mail: shin@glyco.sci.hokudai.ac.jp.

† Dedicated in respect and thankful commemoration to Prof.Dr. F. Cramer who recently deceased three months before his80th birthday.

‡ Ludwig-Maximilians-University Munich.§ Laboratory for Glycocluster Project, Hokkaido University.| Division of Biological Sciences, Hokkaido University.

1 Abbreviations: ASF, asialofetuin; CD, cyclodextrin; Gal,galactose; IgG, immunoglobulin G; Lac, lactose; Lac-BSA, lac-tosylated bovine serum albumin; LacNAc, N-acetyllactosamine;SAP, serum amyloid P component; VAA, Viscum album L.agglutinin.

87Bioconjugate Chem. 2004, 15, 87−98

10.1021/bc0340666 CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 11/22/2003

et al., 2000; Gabius, 2001a; Cloninger, 2002; Housemanand Mrksich, 2002).

Presently, a major challenge in this area is to figureout how spatial factors and binding avidity towardsoluble lectins are correlated. Model studies revealed thatsugar surface density is a crucial factor for selectivity ofbinding, rate of cluster formation, and their stoichiometry(Horan et al., 1999; Cairo et al., 2002). However, in-creases in epitope density in neoglycoconjugates do notnecessarily entail improved capacity as competitiveinhibitors in model systems (Zanini and Roy, 1997a,b;Ashton et al., 1998; Andre et al., 1999a, 2001). Thus,systematic studies are warranted. After the chemistryto graft bioactive carbohydrates onto synthetic/naturalscaffolds had been mastered, first studies on molecularinteractions were nearly exclusively performed with plantlectins. Of note, the behavior of endogenous lectins cannotbe reliably deduced on this experimental basis. As studyobjects, we consequently included mammalian proteinsfrom a lectin family with adhesion/growth-regulatoryactivity on normal and malignant cells, i.e., the galectins(Gabius, 1997; Hirabayashi, 1997; Ohannesian and Lo-tan, 1997; Kaltner and Stierstorfer, 1998; Andre et al.,1999b; Brewer, 2002; Danguy et al., 2002; Rabinovich etal., 2002). For comparison, we also worked with agalactoside-specific plant lectin with potent signalingactivity andsat increased concentrationstoxicity onmammalian cells (Endo, 1989; Timoshenko et al., 1999;Gabius, 2001b) and an affinity-purified human immu-noglobulin G fraction, a model for auto- or xenoreactivecarbohydrate-binding antibodies in serum. Similar togalectins, these two sugar receptors are also cross-linkingmodules (Gupta et al., 1996). Persubstituted â-cyclodex-trins maintaining their Cn-symmetry were chosen as testsubstance owing to several favorable properties forpotential applications.

Cyclodextrins are nonimmunogenic natural macro-cycles with inherently low pharmacological activity andhigh biocompatibility, the capacity to host pharmaceu-ticals in their truncated cone-shaped hydrophobic cavitycoming along as added value. Functionalization withcarbohydrate derivatives was accomplished with the long-term aim to devise an “intelligent” drug delivery system(Fulton and Stoddart, 2001a; Houseman and Mrksich,2002; Ortiz Mellet et al., 2002). Having developed thefacile synthesis of persubstituted â-cyclodextrins withgalactose (Gal), lactose (Lac), or N-acetyllactosamine(LacNAc) as headgroup (Furuike et al., 2000), theseglycoclusters (for schematic illustration, please see Figure1) were introduced to assays with galectins. Theseendogenous lectins harbor marked intrafamily complex-ity. It encompasses different modes of spatial orientationsof the carbohydrate recognition domains and cross-linking capacity, leading to functional divergence indisease mechanisms such as tumor invasion and spread(Kopitz et al., 2001; Lahm et al., 2001; Brewer, 2002;Camby et al., 2002; Cooper, 2002; Nangia-Makker et al.,2002; Nagy et al., 2003). For this study, we selected tworelated prototype proteins (galectins-1 and -7) and thechimera-type galectin-3. Because sugar density andtopology are emerging as crucial factors modulating lectinbinding, we systematically tested the glycoclusters’ ca-pacity to inhibit lectin binding to a panel of surface-presented ligands. Toward this end, (neo)glycoproteinspresenting either lactose maxiclusters, bi- or triantennaryN-glycans, or a complex mixture of N- and O-glycanswere used for establishing the model surface. Last butnot least, our study design accounted for another param-eter of importance: the potency to interfere with multi-

valent interactions can vary with the nature of the testsystem (Mann et al., 1998). To address this issue prop-erly, we performed solid-phase, haemagglutination, andcell binding assays. In these experimental settings thelectins were invariably present in solution. To be able toprobe galectin behavior when presented on a cell surface,we generated a stable galectin-1-overexpressing colorec-tal cancer cell clone by transfection as a test model. Thepresented data reveal (a) efficiency of the substitutedcyclodextrins to abolish receptor binding, (b) differentialsensitivity of the tested galectins, (c) the improtance ofligand topology, and (d) of lectin presentation either insolution or on the cell surface.

EXPERIMENTAL PROCEDURES

Reagents. The persubstituted glycoclusters with ga-lactose, lactose, and N-acetyllactosamine as headgroup(please see Figure 1 for structural details) were preparedby conjugation of the respective sodium thiolate derivedfrom treatment of 3-(3-thioacetyl propionamido)propylsugars with NaOMe to heptakis 6-deoxy-6-iodo-â-cyclo-dextrin at 70 °C for 24 h, as described (Furuike et al.,2000). Following gel filtration on Sephadex G25, concen-tration of peak fractions, washing of the crude solid andlyophilization analytical procedures (optical rotation, 1Hand 13C NMR spectroscopy, and matrix-assisted laserdesorption ionization time-of-flight mass spectrometry)checked structure and purity of the products prior to theinhibition studies. Galectin-1 from bovine heart, murinegalectin-3 (using the plasmid prCBP35s and E. coli JA221 cells; a kind gift of Dr. J. L. Wang), and humangalectin-7 (using the plasmid pQE-60/hGal-7 and E. coliM15[pREP4] cells; a kind gift of Dr. F.-T. Liu), the

Figure 1. Structural illustration of the perglycosylated â-cy-clodextrin derived from coupling 3-(3-thioacetyl propionamido)-propyl glycosides after de-O,S-acetylation to yield unprotectedglycosides with a terminal sodium thiolate to heptakis 6-deoxy-6-iodo-â-cyclodextrin.

88 Bioconjugate Chem., Vol. 15, No. 1, 2004 Andre et al.

galactoside-specific agglutinin from Viscum album L.(VAA) and the lactoside-binding immunoglobulin G frac-tion of human serum, subfractionated by removing anyR-galactoside-binding activity, were isolated and theirpurity was checked by one- and two-dimensional gelelectrophoresis as described (Gabius, 1990; Andre et al.,1997). Biotinylation was performed under optimizedactivity-preserving conditions, and the binding partnersfor the sugar receptors, i.e., lactosylated bovine serumalbumin with an average ligand density of 28 moietiesper carrier molecule, human serum amyloid P compo-nent, and bovine asialofetuin, were prepared, as de-scribed previously (Andre et al., 2001). Murine Engelbreth-Holm-Swarm laminin was a kind gift by Dr. R. Timpl.

Quaternary Structure Analysis. Aliquots of 100 µgof galectin were run over a prepacked Superose 12 HR10/30 column (24 mL bed volume) connected to a HPLCsystem (Hitachi-Merck, Darmstadt, Germany) with 50mM phosphate-buffered saline (pH 7.2) without/with 100mM lactose at a flow rate of 0.7 mL/min. Commercialstandard proteins (BioRad, Munich, Germany) were usedfor calibration.

Inhibition Assays. The solid-phase assays were per-formed in microtiter plate wells (Microlon; Greiner,Nurtingen, Germany). Surface coating of the (neo)-glycoproteins was carried with an optimal concentrationbased on initial systematic testing (please see Table 1and Table 2 for details) with 50 µL of 20 mM phosphate-buffered saline (pH 7.2) overnight at 4 °C. Thoroughwashing, blocking of remaining protein-binding sites with100 µL of buffer solution containing 1% carbohydrate-free bovine serum albumin for 1 h at 37 °C and againthorough washing preceded the incubation step with thebiotinylated sugar receptor in the absence/presence ofglycocluster for 1 h at 37 °C. This mixture had beenpreincubated for 30 min at 37 °C without visible precipi-tate formation. Signal generation by streptavidin-per-oxidase and o-phenylenediamine/H2O2 and its quantita-tion followed, as described in detail (Andre et al., 1997).Carbohydrate-dependent haemagglutination of trypsin-treated, glutaraldehyde-fixed rabbit erythrocytes by lec-tins was monitored with increasing concentrations ofglycoinhibitor to determine the minimal inhibitory con-centration, as described (Gabius et al., 1984). Flowcytofluorimetric analysis of binding of labeled galectinsor lactosylated neoglycoprotein to tumor cell surfaces(human colon adenocarcinoma line SW480 obtained from

the American Type Culture Collection, Rockville, MD,and human B-lymphoblastoid line Croco II establishedand propagated as described by Gabius et al., 1991) usedstandard FACScan instrumentation (Becton-Dickinson,Heidelberg, Germany, equipped with the software pack-age CellQuest Pro) and fluorescent streptavidin-R-phy-coerythrin (Sigma, Munich, Germany) as signal-gener-ating reagent, as described (Kojima et al., 1997).

Generation of Stable Galectin-1-OverexpressingTransfectants. Cells of the human colorectal adenocar-cinoma line DLD-1 (American Type Culture Collection,Rockville, MD) were grown in RPMI 1640 medium with10% fetal calf serum (Biochrom, Berlin, Germany), 2 mML-glutamine, 100 U/mL penicillin, and 100 µg/mL strep-tomycin. For transfection full-length cDNA for humangalectin-1, kindly provided in the pH14Gal vector by Dr.J. Hirabayashi, was subcloned into the Kpn I restrictionsite of the pcDNA3.1(+) vector (Invitrogen/Life Technolo-gies, Karlsruhe, Germany). Using 60-80% confluentcultures and the SuperFect reagent (Qiagen, Hilden,Germany; 10 µL/100 µL serum-free medium × 2 µg vectorDNA) the cells were transfected and selection with G418(600 µg/mL) initiated. Mock transfection with insert-free(null) vector was performed in parallel.

Characterization of Stable Transfectants. Inser-tion of the cDNA-carrying vector into genomic DNA wasverified by PCR. Pellets from 2 × 106 cells were treatedwith 600 µL of nuclear lysis solution (Promega, Mann-heim, Germany) and RNase. Following precipitation ofproteins the genomic DNA in solution was pelleted byaddition of 2-propanol and centrifugation. A 0.5 µgamount of this material was subjected to PCR analysisusing the following pair of sense (5′-CGCTAGGGTAC-CATGGCTTGTGGTCTGGTCG-3′) and antisense (5′-CG-TACGGGTACCTCAGTCAAAGGCCACACA-3′; KpnI siteunderlined) sequences. In parallel, actin gene amplifica-tion was run. For the RT-PCR analysis total RNA wasextracted with the RNAeasy kit reagents (Quiagen,Hilden, Germany) and reverse transcription used 200 USuperscript II RNase H- Reverse Transcriptase (Invit-rogen/Life Technologies, Karlsruhe, Germany). Westernblotting analysis from total cell extracts with non-cross-reactive anti-galectin-1 antibody (1 µg immunoglobulinG/ml) was performed, as described (Gabius et al., 1986;Kaltner et al., 2002). Specifically bound antibody wasvisualized with an horseradish peroxidase/goat anti-rabbit immunoglobulin G conjugate (0.2 µg/mL) and a

Table 1. Determination of the IC50 Values and the Inhibitory Capacity (relative potency, rel pot.) of Gal-, Lac-, andLacNAc-Containing â-Cyclodextrins Relative to the Univalent Inhibitor Lactose in a Solid-Phase Assay

Lac-BSA SAP ASF laminin

inhibitorsugar content/

molecule IC50 (µM) rel pot. IC50 (µM) rel pot. IC50 (µM) rel pot. IC50 (µM) rel pot.

A. With Surface-Immobilized (Neo)glycoproteins and VAA in Solutiona

D-gal 1 2500 0.4 20000 0.38 5000 0.6 900 0.56lactose 1 1000 1 7500 1 3000 1 500 1Gal-CD 7 4.9 204 (29.1)b 48.7 154 (22.0) 122 24.6 (3.5) 36.5 13.7 (2.0)Lac-CD 7 0.45 2222 (317) 181 41.4 (5.9) 2.3 1304 (186) 2.7 185 (26.4)LacNAc-CD 7 25.5 39.2 (5.6) 255 17.6 (2.5) 212 ) 44% inhib <14.5 (<2.1) 53.1 9.4 (1.3)

B. With Surface-Immobilized (Neo)glycoproteins and Lactoside-Binding IgG in Solutionc

D-gal 1 100000 0.03 200000 1 55000 1.8lactose 1 3000 1 200000 1 100000 1Gal-CD 7 6.1 492 (70.3)d 183 1093 (156) 36.5 2740 (391)Lac-CD 7 15.8 190 (27.1) 904 ) 22.8% inhib <221 (<31.6) 452 221 (31.6)LacNAc-CD 7 6.4 469 (67.0) 425 471 (67.3) 12.8 781 (112)

a The assays were performed at the following combinations of lectin and (neo)glycoprotein concentration: VAA/Lac-BSA (1.5 µg/mL/0.05 µg), VAA/SAP (1.5 µg/mL/0.5 µg), VAA/ASF (1.5 µg/mL/1µg), VAA/laminin (3 µg/mL/0.5 µg/mL). b The numbers in parentheses denotethe relative potency of each sugar unit in the heptavalent cyclodextrin compared to carrier-free lactose set as internal quality standardc The assays were performed at the following combinations of IgG and (neo)glycoprotein concentration: Lac-IgG/Lac-BSA (1 µg/mL/0.05µg), Lac-IgG /SAP (10 µg/mL/0.5 µg), Lac-IgG /ASF (10 µg/mL/1µg); laminin was not tested as matrix. d The numbers in parenthesesdenote the relative potency of each sugar unit in the heptavalent cyclodextrin compared to carrier-free lactose set as internal qualitystandard.

Inhibition of Lectin Binding by Glycoclusters Bioconjugate Chem., Vol. 15, No. 1, 2004 89

chemiluminescence kit system (Amersham/PharmaciaBiotech, Freiburg, Germany). In a second round ofprobing actin was detected on the same blots by apolyclonal anti-actin antibody (Sigma, Munich, Germany;3 µg/mL). Cell growth kinetics was analyzed for 10 daysstarting with a cell density of 104 cells/ml in 24-wellculture plates loaded with 1 mL at 24 h intervals usingan improved Neubauer chamber. Flow cytofluorimetricdetection of cell surface galectin-1 was carried out, asdescribed above for cell binding of lectins and lactosylatedneoglycoprotein using a goat anti-rabbit immunoglobulinG FTC-conjugate (Sigma, Munich, Germany; 1:100 dilu-tion of commercial solution).

RESULTS

Quaternary Galectin Structure. The three testedmammalian galectins belong to different subgroupswithin this family of endogenous lectins. Galectins-1 and-7 are prototype family members. They are characterizedby the presence of a protomer harboring one carbohydraterecognition domain. In principle, it may or may notassociate noncovalently to a dimeric cross-linking module.Galectin-3, in contrast, is the only known chimera-typefamily member. Its C-terminal carbohydrate recognitiondomain is linked to a collagenase-sensitive tandem-repeatregion and a short N-terminal stretch. To ascertain thequaternary structure of these preparations of galectins-1and -3 in solution, we performed molecular weightanalysis by analytical gel filtration. Using a Superosematrix containing galactose and 3,6-anhydro-L-galactosein its repeating unit (agarobiose), the elution profile forgalectin-1 confirmed its complete dimerization, whereasgalectin-3 was retarded considerably in relation to the

expected position (Figure 2, upper panel). Recallingbinding capacity of galectin-3 to both nonreducing ter-minal and internal LacNAc sequences (Ahmad et al.,2002), a weak carbohydrate-specific interaction with thematrix was a reasonable and testable explanation. Wetherefore repeated the analysis in the presence of 100mM lactose to block it. Indeed, the elution peak ofgalectin-3 was shifted to the position of the monomer(calculated MW: 27,384 Da) as predicted (Figure 2,bottom panel). This result underscored the necessity forrigorous specificity controls in the binding assay espe-cially for galectin-3 (please see below).

With these two positions in the elution profile extend-ing the set of molecular weight markers, we next ad-dressed the question on the quaternary structure ofhuman galectin-7. To preclude that weak and transientbinding to the matrix might erroneously intimate pres-ence of a monomer, this analysis was likewise performedin the absence and presence of lactose. Irrespective ofaltering this parameter galectin-7 eluted exclusively atthe position of the dimer like galectin-1 (Figure 2). Thisresult is in accord with recent mass spectrometric as-sessment (Kopitz et al., 2003). Galectin-7 is thus adimeric prototype galectin in solution. It is noteworthythat its retention time (18.8 min) was longer than thatfor galectin-1 (16.52 min) despite a molecular weightdifference of 546 Da in favor of galectin-7. Evidently, theshape of the two prototype galectins differed in solution.This intragroup variation is a strong argument to includemembers of the same subclass into our investigationwhether and to what extent the tested multivalentinhibitors will affect lectin binding. Two principal typesof inhibition assay were performed to answer this ques-

Table 2. Determination of the IC50 Values and the Inhibitory Capacity (relative potency, rel pot.) of Gal-, Lac-, andLacNAc-Containing â-Cyclodextrins Relative to the Univalent Inhibitor Lactose in a Solid-Phase Assay

Lac-BSA SAP ASF laminin

inhibitor

sugarcontent/molecule IC50 (µM) rel pot. IC50 (µM) rel pot. IC50 (µM) rel pot. IC50 (µM) rel pot.

A. With Surface-Immobilized (Neo)glycoproteins and Galectin-1 in Solutiona

D-gal 1 40000 0.03 200 mM )20% inhib

70000 0.06 200 mM )6% inhib

lactose 1 1100 1 180000 1 4000 1 300000 1Gal-CD 7 30.5 36.1 (5.2)b 1218 )

0% inhib609 )

11% inhib< 6.6 (<0.9) 1218 )

32.2% inhibLac-CD 7 54.2 20.3 (2.9) 904 )

8.7% inhib226 )

33% inhib< 17.7 (<2.5) 904 )

0% inhibLacNAc-CD 7 63.8 17.2 (2.5) 850 212 (30.3) 425 9.4 (1.3) 850 )

0% inhib

B. With Surface-Immobilized (Neo)glycoproteins and Galectin-3 in Solutionc

D-gal 1 60000 0.01 30000 0.03 55000 0.02 15000 0.03lactose 1 500 1 800 1 1000 1 400 1Gal-CD 7 1218 )

0% inhib<0.4 (<0.1)d 609 )

8.9% inhib<1.3 (<0.2) 1218 )

37.1% inhib<0.82 (<0.1) 609 )

36.3% inhib<0.7 (<0.1)

Lac-CD 7 9.0 55.6 (7.9) 13.6 58.8 (8.4) 181 5.5 (0.8) 113 3.5 (0.5)LacNAc-CD 7 149 3.4 (0.5) 53.1 15.1 (2.2) 85 11.8 (1.7) 34.0 11.8 (1.7)

C. With Surface-Immobilized (Neo)glycoproteins and Galectin-7 in Solutione

D-gal 1 35000 0.14 95000 0.06 4500 0.18 15000 0.13lactose 1 5000 1 6000 1 800 1 2000 1Gal-CD 7 1218 )

37% inhib<0.8 (<0.1)f 1218 )

4.8% inhib<4.9 (<0.7) 1218 )

34% inhib0.66 (0.1) 1218 mM )

5.7% inhib<1.6 (<0.2)

Lac-CD 7 452 2.2 (0.3) 904 6.6 (0.9) 181 4.4 (0.6) 452 4.4 (0.6)LacNAc-CD 7 425 2.4 (0.3) 850 )36.9% inhib <7.1 (<1.0) 383 2.1 (0.3) 850 )

38.1% inhib<2.4 (<0.3)

a The assays were performed at the following combinations of lectin and (neo)glycoprotein concentration: galectin-1/Lac-BSA (10 µg/mL/1 µg), galectin-1/SAP (15 µg/mL/0.5 µg), galectin-1/ASF (10 µg/mL/1µg), galectin-1/laminin (10 µg/mL/0.5 µg/mL). b The numbers inparentheses denote the relative potency of each sugar unit in the heptavalent cyclodextrin compared to carrier-free lactose set as internalquality standard. c The assays were performed at the following combinations of lectin and (neo)glycoprotein concentration: galectin-3/Lac-BSA (5 µg/mL/0.25 µg), galectin-3/SAP (10 µg/mL/0.5 µg), galectin-3/ASF (5 µg/mL/1µg), galectin-3/laminin (20 µg/mL/0.5 µg/mL).d The numbers in parentheses denote the relative potency of each sugar unit in the heptavalent cyclodextrin compared to carrier-freelactose set as internal quality standard. e The assays were performed at the following combinations of lectin and (neo)glycoproteinconcentration: galectin-7/Lac-BSA (30 µg/mL/0.05 µg), galectin-7/SAP (30 µg/mL/0.5 µg), galectin-7/ASF (30 µg/mL/1µg), galectin-7/laminin(30 µg/mL/0.5 µg/mL). f The numbers in parentheses denote the relative potency of each sugar unit in the heptavalent cyclodextrin comparedto carrier-free lactose set as internal quality standard.

90 Bioconjugate Chem., Vol. 15, No. 1, 2004 Andre et al.

tion: a.) solid-phase binding to a matrix of biochemicallydefined (neo)glycoproteins mimicking a standardized cellsurface and b.) cell-based binding using erythrocytes aslaboratory models as well as cultured tumor cells and alectin-overexpressing cell clone after gene transfection forclinical relevance. We tested persubstituted cyclodextrinsto assess their potency to interfere with the protein-carbohydrate interactions.

Cyclodextrins as Inhibitors of Binding to (Neo)-glycoproteins. The adsorption of (neo)glycoproteins tothe surface of ELISA plate wells established a matrixwith ligand properties of a cell surface. The way thepersubstituted â-cyclodextrins meddle with lectin bindingto the glycans of the artificial glycocalyx can thus beconsidered as model situation to measure their influenceon extracellular lectin activities. In the first step, thesugar dependence of the interaction between the surface-immobilized (neo)glycoproteins and the sugar receptorswas ascertained. Invariably, binding for all sugar recep-tors was saturable and sensitive to presence of thehaptenic sugar but not mannose. We performed ourmeasurements with lactosylated albumin, presentingrather widely spaced ligand sites (maxiclusters), andthree glycoproteins with known differences in glycanfeatures: the human pentraxin serum amyloid P com-ponent (SAP) with a single complex-type biantennaryN-glycan at Asn32 per protomer which -convenient forinteraction studies - extends outward from the subunits,asialofetuin with preferentially triantennary N-glycansat its three N-glycosylation sites and murine lamininharboring a complex array of especially bi- and trianten-nary N-glycans including poly(N-acetyllactosamine) elon-gations distributed over 73 sites for N-glycosylation.

Thus, this set enabled us to relate inhibitory capacity tothe lectin and also to a defined glycan display, the latterparameter so far not thoroughly evaluated. To excludethat the cyclooligosaccharide carrier itself impairedreceptor binding, we performed assays to figure out asto whether presence of unsubstituted â-cyclodextrinsaffected extent of binding of the biotinylated markers.Even at the very high concentration of 3.52 mM no oronly minor (up to 15%) inhibitory activity could generallybe picked up under the standard assay conditions.Looking at the data reported in the first section ofResults, an expectable exception was galectin-3, with thisconcentration for example reaching 43.4% inhibition ofbinding to SAP. Because functionalization improved theinhibitory capacity of the cyclooligosaccharide by up to 2orders of magnitude, these effects could yet be consideredto be minor. Maintaining the overall symmetric geometryof their presentation, we attached three different head-groups to the carrier scaffold, i.e., Gal, Lac, and LacNAcvia the same linker structure. Thus, we could determineexclusively the influence of headgroup tailoring underotherwise identical conditions. â-Cyclodextrin-based gly-coclusters were then tested as competitive inhibitors tomatrix binding of the sugar receptors kept in solution.

By raising the concentration of the glycocluster in theindividual experimental series, the percentage of surface-bound receptor was reduced to varying extents, asexemplarily shown in Figure 3. Conjugation of thecarbohydrate derivatives to the cyclodextrin scaffold thusdid not harm binding properties of the headgroups tolectins and immunoglobulins. To calculate the glycoclus-ters‘ efficiency, we set an internal standard by runningassays with galactose and lactose as reference substancesin parallel. Tables 1 and 2 present the data for the fivereceptor types and the four (neo)glycoproteins as IC50values. The measurements revealed that the glycoclus-ters had pronounced activities, the immunoglobulin Gfraction and the plant lectin being markedly responsive(Tables 1a,b). Importantly, the topological display of theglycans (maxicluster, bi- or triantennary N-glycan chains)of the surface-presented ligand mattered. Plant lectinbinding to maxiclusters and triantennary glycans waspreferentially sensitive to glycocluster presence (Table1a). Galactose-presenting scaffolds impaired binding ofthe serum immunoglobulin G fraction, too, especially inthe presence of triantennary N-glycans of ASF in theartificial glycocalyx (Table 1b). Further synthetic ma-nipulations of the glycoclusters by adding a glucosemoiety and then a N-acetyl group were apparently notbeneficial in this case. Regarding the galectins, there is

Figure 2. Determination of the molecular weight under nativeconditions for bovine galectin-1 (G1, arrow), murine galectin-3(G3, arrow), and human galectin-7 (elution profile) with aSuperose 12 HR 10/30 column. The analysis was performed inrunning buffer without addition of lactose (top) and in thepresence of 100 mM lactose (bottom). Positions of molecularweight markers for calibration are given by arrowheads: bovineimmunoglobulin (1) at 159 kDa, bovine serum albumin (2) at66 kDa, carbonic anhydrase (3) at 29 kDa, cytochrome c (4) at12.4 kDa, and aprotinin (5) at 6.5 kDa.

Figure 3. Inhibition curves of binding of biotinylated galac-toside-specific Viscum album L. agglutinin to surface-immobi-lized murine laminin using the â-cyclodextrin-based glycoclus-ters with derivatives of D-galactose (0), lactose (b) andN-acetyllactosamine (2) as headgroup.

Inhibition of Lectin Binding by Glycoclusters Bioconjugate Chem., Vol. 15, No. 1, 2004 91

a general trend that headgroup extension improved theinhibitory efficiency (Table 2a-c). However, the overallsensitivity did often not reach the high level of inhibitiondetected for the other two sugar receptors. Among thegalectins, galectin-3 showed the comparatively bestresponsiveness (Table 2b). In comparison, the two pro-totype proteins were generally not very reactive withthese glycoclusters, although interindividual differenceswere apparent between galectins-1 and -7 (Table 2a-c).The inhibition of galectin-3 binding to maxiclusters andbiantennary N-glycans showed a stronger dependence oninhibitor multivalency than that to the other two glyco-proteins (Table 2b vs Table 2a,c). When calculating therelative potency of each carbohydrate unit of the cyclicglycoclusters, rather modest increases were obtained forthe prototype galectins, depending on the nature of theligand tested, relative to the other two sugar receptortypes (Tables 1a,b, Tables 2a,c). Because a potential fieldof application will entail blocking binding to cell surfaces,not a matrix established by a single (neo)glycoprotein,we next tested the glycoclusters in assays using differentcell types.

Cyclodextrins as Inhibitors of Binding to Cells.In the first test system we determined the extent ofinhibition of lectin-mediated haemagglutination usingtrypsin-treated, glutaraldehyde-fixed rabbit erythrocytesas popular cell model. The agglutination was sugardependent and not affected by the unsubstituted cyclo-dextrin up to 3.5 mM, as shown by controls (Table 3).The glycoclusters were clearly inhibitory, especially forthe plant lectin. In this case, their design led to animprovement of the inhibitory capacity on the level ofthe individual sugar molecule by the factor of 11.8 indirect comparison to free galactose (Table 3). Corroborat-ing the results of the solid-phase assays, the bridgingactivity of galectins was also inhibited by the function-alized macrocycles, especially with LacNAc as headgroup.Relative to the plant lectin, their activity toward thetested prototype galectin again was less pronounced. Onthe other hand, galectin-3, a weak haemagglutinin dueto the requirement of aggregation when surface boundas it is monomeric in solution (please see Figure 1), wasa rather suitable target (Table 3). To address the issueas to whether the assay design using cell agglutinationand not cell binding as endpoint might influence theresults, we performed flow cytofluorimetric measure-ments with two tumor cell lines (the B-lymphoblastoidline Croco II and the colon carcinoma line SW480). Wetested the glycoclusters as inhibitors of lectin binding tothe cell surface.

The results clearly showed that both assay typesmonitoring cell binding (haemagglutination or surfacebinding of labeled lectin) yielded comparable results. As

shown in Figure 4 for the Croco II cells, the functionalizedâ-cyclodextrin was a potent inhibitor for the plant lectinrelative to the monovalent sugar (top panel). The inhibi-tion of galectin-3 binding by the Lac/LacNAc-bearingglycoclusters was significant and in the same favorablerange, which had been monitored in haemagglutination,compared to the free sugar (middle and bottom panels).Up to this point, the assays were designed with a ligand-exposing surface to which the receptor coming fromsolution associates. The inhibitor and the receptor werethus both in solution. This situation simulated extracel-lular activities of galectins. In addition and equallyimportant, they can also exert functions from the cellsurface. To further define the extent to which theglycoclusters inhibit interactions mediated by cell surfacelectins, it was thus essential to determine their influenceon blocking galectin functionality on the cell surface. Atthis site, the lectin such as galectin-1 can act as a signalsensor or as a cell migration/adhesion molecule directingtransient or firm adhesion to other cells and/or extracel-lular matrix glycoproteins, a step crucial for metastasis.To assign inhibitory potency of the glycoclusters onbinding of glycans to a distinct galectin of the cell surface,a tumor cell line with strong surface expression of acertain galectin was required. The two tumor lines usedabove produce a set of four galectins, namely types 1, 3,8, and 9 (Gabius et al., 1991; Lahm et al., 2001),confounding the interpretation. For this purpose, wegenerated a clone by gene transfection, using the humancolorectal cancer line DLD-1 and galectin-1 as models.

Stable gene transfection by a galectin-1-cDNA-bearingexpression vector and ensuing galectin-1 overexpressionon the mRNA and protein levels were verified by PCRanalysis of genomic DNA, RT-PCR monitoring, andWestern blotting (Figure 5). The primer sequences werechosen to yield an amplification product of 408 bp fromthe vector-based cDNA in the genomic DNA and themRNA. The length of the amplification product from theregular galectin-1 gene in the genomic DNA is 8142 bp.Highly efficient reverse transcription and Western blot-ting revealed traces of galectin-1-specific products in wild-type cells, signal absence in mock-transfected cells, andvery strong expression in the sense-transfected cells. Themorphological appearance of the three cell populationswas rather similar (Figure 6), excluding a detrimentaleffect caused by this genetic manipulation. Also, the cellgrowth kinetics of the cell populations gave no indicationsfor a markedly altered behavior with impaired membranefunctions (Figure 7). Of interest for our study was themeasurement of cell surface presentation of galectin-1.Relative to the control value the anti-galectin-1 antibodybinding to the cell surface was clearly increased for thestably transfected cells (Figure 8). Using fluorescent

Table 3. Determination of the Minimal Inhibitory Concentration (MIC) and the Relative Inhibitory Capacity ofGlycosubstances in Lectin-Mediated Haemagglutination

VAA (0.1 µg/mL) galectin-1 (1 µg/mL) galectin-3 (100 µg/mL)

inhibitor MIC [µM] relative potency MIC [µM] relative potency MIC [µM] relative potency

D-galactose 25000 0.5 6250 0.1 50000 0.06lactose 12500 1 625 1 3000 1ASF 10.5 1190 (132)a 2.5 250 (27.8) 105 28.6 (3.2)â-CD no inhibition

up to 3500 µM<3.6 no inhibition

up to 3500 µM<0.18 no inhibition

up to 3500 µM<0.86

Gal-CD 305 41.0 (5.9) 1218 0.5 (0.07) no inhibitionup to 1218 µM

< 2.5

Lac-CD 452 27.7 (4.0) 226 2.8 (0.4) 452 6.6 (0.9)LacNAc-CD 850 14.7 (2.1) 106 5.9 (0.8) 106 28.3 (4)

a The numbers in parentheses denote the relative potency of each sugar unit in the heptavalent cyclodextrin compared to carrier-freelactose set as internal quality standard.

92 Bioconjugate Chem., Vol. 15, No. 1, 2004 Andre et al.

lactosylated albumin as probe, the inhibition of its cellsurface association by lactose and lactose-bearing â-cy-clodextrin was compared in FACScan analysis. Notably,the maxiclusters on the neogycoprotein enabled multi-valent contacts to the surface-presented lectin, as alreadyshown for several tumor cell lines and neoglycoenzymesas probes (Gabius et al., 1990). With the lactose concen-tration set at 6.4 mM, the percentage of positive cells wasdecreased to 70.5% relative to the control. The â-cyclo-dextrin derivative used at the same concentration alsoreduced binding of the neoglycoprotein to the cells, 59.5%of the cells remaining positive. Remarkably, even anincrease of the free lactose concentration to 200 mM didnot reach this level of competition with multivalent

binding. The same tendency was apparent for the meanfluorescence intensity of cells. This result indicates thatthis galectin was more responsive to the multivalentinhibitor when presented on the cell surface than whenin solution.

DISCUSSION

A general feature of protein-carbohydrate interactionsis the requirement for establishment of several contactsto generate a sufficiently high affinity for biologicalactivities. Therefore, carbohydrate recognition domainsin lectins are often clustered, as recently summarized andillustrated for mammalian lectins (Gabius et al., 2002).A panel of structural motifs gives rise to diverse waysfor the relative orientation of the binding sites. “Intel-ligent“ blocking reagents or drug-delivery systems willcapitalize on this factor to selectively home in on theirtargets. Advances in synthetic chemistry to solve prob-lems of convenient carrier functionalization have madeit possible to move from the preparative phase to testingthe custom-made glycoclusters. In the case of hepta-antennated â-cyclodextrins, plant lectins have so farserved as model receptors. Respective measurements ledto the conclusions that multivalency triggered nonuni-form responses for various agglutinins in inhibitionassays and that an increase in sugar density will notnecessarily entail linear enhancement of inhibitory ca-pacity.

In detail, galactosyl clusters interacted with the peanutlectin leading to a 16-fold increased association constantrelative to the monovalent analogue but the individualligands in two forms of persubstituted â-cyclodextrins (i.e.heptakis[6-S-â-D-galactopyranosyl-6-thio]cyclomaltohep-taose and heptakis[6-amino-6-deoxy-6-N-(â-D-galacto-pyranosyl-1-thiomethyl-carbonyl]cyclomaltoheptaose) failedto retain the level of inhibitory activity of a galactosemoiety as internal standard in a solid-phase assay(Garcıa-Lopez et al., 1999; Yasuda et al., 2000). The sameinhibitor design gave reactivity for the pea lectin, theGriffonia simplicifolia I and wheat germ agglutinins butnot the lectin from Galanthus nivalis (Ichikawa et al.,2000; Yasuda et al., 2000). Wheat germ agglutinin wasalso found to be remarkably responsive, whereas thedimeric Erythrina corallodendron lectin showed a re-duced level of sensitivity (Furuike et al., 2000). Interest-ingly, studies with this lectin or its closely related variantfrom Erythrina cristagalli had not detected a strong effectof multivalency in dendrimers or neoglycopolymers (Za-nini and Roy, 1997a; Pohl and Kiessling, 1999). This bodyof evidence discourages prediction of a priori potentactivity of glycoclusters on lectins, although they harbormore than one binding site (Rudiger and Gabius, 2001;Loris, 2002). It rather advises consideration of systematicexperimental work as mandatory. Moving from hepta-to tetradeca-antennated glycoclusters, the apparent bind-ing affinity to concanavalin A was not improved and, ofrelevance for drug targeting approaches, the ability ofthe cyclodextrin carrier to form an inclusion complex with2-naphthalenesulfonate was harmed (Ortega-Caballeroet al., 2001). This behavior is evocative of the eventualdecrease in inhibitory capacity of Tris-branched dendriticwedges attached by 1,3,5-trisubstitution to a benzenoidcore by increasing the number of headgroups from nine,then 18 to finally 36 carbohydrate residues (Ashton etal., 1998). The way the nature of the linker can evendrastically influence the inhibition profile was illustratedin the case of heptasubstitution of primary hydroxylswith mannopyranosylthioureido moieties which fail tobind to concanavalin A (Baussanne et al., 2001; Ortiz

Figure 4. Semilogarithmic representation of fluorescent stain-ing of the surface of human B-lymphoblastoid Croco II cells bybiotinylated lectins in the absence and in the presence ofglycoinhibitors using streptavidin R-phycoerythrin as second-step reagent. Top panel: cell staining after incubation with thesecond-step reagent only (gray line), with 0.5 µg mistletoe lectin/ml in the absence of any inhibitor (gray area; left) and in thepresence of 6.4 mM lactose (black line; left), 3.5 mM â-cyclo-dextrin (gray area; right) and 0.9 mM lactose-presenting â-cy-clodextrin (equivalent of 6.3 mM lactose in the heptavalentglycoclusters (black line; right)). Middle and bottom panels: cellstaining after incubation with the second-step reagent only (grayline), with 10 µg galectin-3/ml in the absence of any inhibitor(gray area; middle panel) and in the presence of 2.2 mMD-galactose (black line in middle panel; left), 1.6 mM lactose(black line in middle panel; right), 180 µM substituent-freeâ-cyclodextrin and 61 µM galactose-presenting â-cyclodextrin(gray area and black line in bottom panel; left) as well as 43µM N-acetyllactosamine- and lactose-presenting â-cyclodextrin(gray area and black line in bottom panel, right).

Inhibition of Lectin Binding by Glycoclusters Bioconjugate Chem., Vol. 15, No. 1, 2004 93

Mellet et al., 2002). When extending the linker’s lengthto give the supposedly required degree of conformationalfreedom, as for example documented by Garcıa-Lopez etal. (1999), it should not be overlooked that long alkylspacer arms might find access to the hydrophobic cavityand that carbohydrates might form interresidual hydro-gen bonds, impairing their accessibility for lectins (Fultonand Stoddart, 2001b). The strong preference of the

mistletoe lectin for lactose relative to N-acetyllactosaminedespite rather similar binding properties in inhibitionassays and isothermal titration calorimetry appears toindicate occurrence of such unfavorable events (Lee etal., 1992; Galanina et al., 1997; Bharadwaj et al., 1999).On the other hand, galectin-1, where presence of theN-acetyl group accounts for a 0.7 kcal/mol increase of-∆G relative to lactose binding (300 K), preferred thisligand in contrast to galectin-7, ∆G in this case decreas-ing from -4.6 for lactose to -4.4 kcal/mol for LacNAc(Ahmad et al., 2002). With this background, Fulton andStoddart (2001a) stated that “it is probably fair toconclude that, in the area of saccharide-branched cyclo-dextrins, and indeed saccharide-branched macrocycles,the research carried out to date only begins to scratchthe surface of understanding the subtleties of the relativespatial orientation of carbohydrate ligands, relative totheir scaffolds and to each other.”

In comparison between the presented reactivity pro-files, the plant lectin with its double trefoil structure(Niwa et al., 2003) was prominent for its sensitivitytoward glycoinhibitor multivalency. Mitogenic effects onimmune and, notably, tumor cells in the ng/mL-range andtoxicity at doses above this concentration, seen in vitrofor cultured and for peripheral blood cells as well ashistotypic tumor cultures (Hajto et al., 1990; Gabius etal., 2001), can thus be abrogated swiftly by these inhi-bitiors. A similar result can be expected for other plantAB toxins such as ricin. Similarly, the natural â-galac-toside-specific immunoglobulin G fraction from humanserum was efficiently blocked by hepta-galactosylatedâ-cyclodextrin. The relative potency on the level of theindividual carrier-attached moiety reached the factor of391 compared to lactose as internal standard. Theremarkable sensitivity of the immunoglobulin to these

Figure 5. Detection of presence of galectin-1-specific cDNA in genomic DNA (left), of galectin-1-specific mRNA by RT-PCR analysis(middle) and of galectin-1 by Western blotting (right) in material obtained from human colorectal adenocarcinoma DLD-1 cells (a )wild type, b: mock-transfected cells, c: galectin-1 sense transfectant). Numbers on the left side of each panel indicate length ofmarkers (M) or molecular weight of standard proteins. Actin-related parameters were determined as positive quality control (bottompanel).

Figure 6. Light micrographs from populations of human wild-type colorectal adenocarcinoma DLD-1 cells (a), a mock-trans-fected variant clone (b) and the galectin-1 sense transfectantclone (c); bar ) 20 µm.

Figure 7. Determination of cell growth of DLD-1 wild-type cells(0), a mock-transfected variant clone (O), and the galectin-1sense transfectant clone (1).

Figure 8. Detection of expression of galectin-1 on the surfaceof wild-type (left) and sense-transfected DLD-1 cells (right) byflow cytofluorimetry (black line) relative to antigen-independentstaining by the fluorescent second-step reagent (gray area) ascontrol.

94 Bioconjugate Chem., Vol. 15, No. 1, 2004 Andre et al.

glycoclusters and also to starburst/wedgelike glycoden-drimers and neoglycopolymers might find practical ap-plication to protect patients from antibody-dependenthyperacute rejection originating from the immunoreac-tivity of carbohydrate xenoantigens (Andre et al., 1999a,2001; Wang et al., 1999; Baek and Roy, 2002).

In contrast to these two sugar receptors, the prototypegalectins with their binding sites on opposing sides of thedimer at a distance of close to 50 Å were less reactive.This result is in line with previous measurements as-saying divalent ligands connected by varying linkerlengths as well as starburst and wedgelike glycoden-drimers (Lee et al., 1990; Andre et al., 1999a, 2001, 2003).However, our measurements with the gene-transfectedcell clone revealed that cell surface presentation canrender the prototype galectin-1 more susceptible to amultivalent ligand compared to its behavior in solution.As the koff-rates from p-aminophenyl lactoside differedonly slightly between the plant lectin and galectin-1 inplasmon resonance monitoring (Dettmann et al., 2000),the impact of the topological aspect is underscored. Also,the differential activity of the substituted â-cyclodextrinsagainst the two prototype galectins, which are bothdimeric, can be interpreted to be a sensitive sensor forslight differences in their shape. They might have abearing on galectin functionality by ligand cross-linkingand thus contribute to explain differences in the biologicalresponse profile of related galectins (Camby et al., 2001;Kopitz et al., 2001; Sheikholeslam-Zadeh et al., 2001;Brewer, 2002; Cao et al., 2002; Nagy et al., 2002, 2003;Timoshenko et al., 2003). It is thus useful to have areliable marker in hand to probe these characteristicscomparatively.

The most notable difference between the galectinsconcerned galectin-3 relative to galectins-1 and -7. Mon-omeric in solution, the chimera-type galectin can formaggregates via its N- and C-terminal domain which areresponsive toward multivalent ligands (Andre et al., 2001and references therein). The relative potency of thesubstituted â-cyclodextrins to galectin-3 distinguishedthis family member from the prototype galectins, a clearindication for subgroup selectivity. This result is in linewith the activity profile of wedgelike glycodendrimers,especially after spacer rigidification (Andre et al., 2001;Vrasidas et al., 2003). The answer to the question as towhether persubstitution is indispensable for this featurecan be figured out by testing less than hepta-antennatedproducts obtained by running conjugation with a sub-saturating concentration of the carbohydrate derivative.Combining these topological factors, i.e., ligand displayand spacer properties, with exploitation of the differencesin carbohydrate fine-specificity, among others the 12-folddifference of inhibitory capacity of R2,6-sialylated diLac-NAc to galectin-3 vs galectin-1 (Ahmad et al., 2002;Hirabayashi et al., 2002), the discriminatory power issure to be further enhanced. In this area, combinatorialsynthesis of carbohydrates and mimetics thereof is likelyto be instrumental to extend the panel of structures,efficiently homing in on a certain lectin (Nishimura, 2001;Ramstrom et al., 2002).

A further salient finding of this report is the influenceof the topology of the lectin’s binding partner on theinhibitory profile. To avoid missing this “topological”specificity, it will be essential to routinely test more thanone biochemically defined ligand. By using (neo)glyco-proteins with precisely known glycan profile (maxiclus-ters, bi- and/or triantennary N-glycans), we obtained anonuniform response to the substituted â-cyclodextrinsfor each tested sugar receptor. Thus, we can in fact

conclude that the characteristics of the ligand population(cluster formation, status of branching) matter more thancurrently appreciated for receptors in solution. Avidityof binding to branched and/or clustered ligands encom-passes both a contact and a topology dimension. Withchemoenzymatically synthesized glycan chains becomingavailable, systematic studies of this aspect are clearlywarranted. The way the natural N-glycan substitutionby core fucosylation modulates lectin affinity to thegalactoside termini of complex-type biantennary N-glycans has recently set a precedent for further effortsin this respect (Unverzagt et al., 2002).

In conclusion, this report proves that hepta-antennatedâ-cyclodextrins present their carbohydrate appendagesin bioactive form for galactoside-specific plant and,notably, mammalian lectins as well as immunoglobulinG. Furthermore, discriminatory potency of these glyco-clusters between two prototype and between prototypevs chimera-type galectins was evidenced. Beside thetopological aspect of binding site arrangement in thereceptor part, the geometry of the ligand structure wasshown to exert a considerable influence, a result withevident biological implications. In more general terms,our results provide a promising example that substitutedâ-cyclodextrins, owing to their favorable pharmacologicproperties, have potential to find their place in the designof “intelligent“ blocking reagents.

ACKNOWLEDGMENT

The skilfull technical assistance of B. Hofer, the kindgifts of reagents from Drs. J. Hirabayashi, F.-T. Liu, R.Timpl, and J. L. Wang, the helpful advice from Drs. B.B. and S. Namirha, and the generous financial supportof the Wilhelm Sander-Stiftung (Munich, Germany) aregratefully acknowledged.

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